Single-chain insulin analogs (mini-proinsulins; SCIs) are of complementary interest as a model system for studies of protein design and as a therapeutic platform for the treatment of diabetes mellitus. This application focuses on an SCI containing a foreshortened C domain (six residues) whose design incorporates novel features of translational interest. Preliminary studies have established that this analog (designated SCI- biphasic) exhibits extraordinary stability at elevated temperatures and has biphasic pharmacodynamics properties similar to those of pre-mixed regular/NPH analog products (Lilly Pre-Mixed 75/25 Insulin lispro or Novo-Nordisk Pre-mixed 70/30 Insulin Aspart). Despite the complexity of their formulation, such pre-mixed products provide Type 1 and Type 2 diabetes patients with a simplified twice-a-day regimen and are in broad use in the developing world. We envisage that an ultra-stable and straightforward soluble formulation of SCI- biphasic would be of humanitarian value in relation to growing prevalence of both Type 1 and Type 2 diabetes. My MD/PhD program of research will focus on the structure, function, and misfolding of SCI-biphasic. Aim 1 employs heteronuclear multidimensional NMR spectroscopy to investigate the structure and dynamics of this insulin analog in solution. We seek to test the hypothesis that specific design elements in the A, B, and C domains contribute to stability and are associated with the damping of conformational fluctuations. Aim 2 focuses on tests of thermal stability, including successive testing of stressed samples in a rat model of diabetes. We seek to test the hypothesis that under the harsh conditions often experienced by underprivileged patients in the developing world (lacking access to electricity or refrigeration) SCI-biphasic will retain potency and biphasic pharmacodynamics for several months whereas current insulin products degrade within days. Aim 3 exploits our serendipitous finding that in the yeast Pichia pastoris (which in rich growth medium efficiently expresses and secretes SCI-biphasic) use of minimal medium leads to the co-secretion of two forms of this analog: one with native disulfide pairing (as in proinsulin and insulin) and the other with a rearrangement of the disulfide bridges. The non-native fold provides a model of the stealth misfolding of a protein in the endoplasmic reticulum (ER) during stress. We seek to identify this non-native isomer and probe its 3D structure as a model for the analogous mispairing and misfolding of proinsulin that occurs in human -cells in the natural history of Type 2 diabetes. Such misfolding has been proposed to contribute to -cell exhaustion in the late stages of this chronic disease. To our knowledge, the structure of this SCI isomer will provide the first visualization of a polypeptide quantitatively inserted into a kinetic trap by the ER oxidative protein-folding machinery.